PROBLEM: Aging is the most important risk factor for Alzheimer's disease (AD), which represents the most common cause of dementia in our country. The disease, for which there is no currently available treatment, is becoming increasingly prevalent among our aging Veteran population. PRELIMINARY DATA: Our group has recently identified a novel form of post-translational regulation that affects both levels and activity of BACE1. Specifically, we discovered that nascent BACE1 is transiently acetylated in the lumen of the ER by two acetyltransferases, which we named ATase1 and ATase2. The acetylated intermediates of nascent BACE1 are able to complete maturation whereas non-acetylated intermediates are rapidly degraded. Consistently, up-regulation of ATase1 and ATase2 increases BACE1 levels and A generation while down-regulation has the opposite effects. Both ATase1 and ATase2 can be detected in a variety of cell lines that are commonly used for the study of the nervous system. In the brain they are preferentially expressed in neurons. Finally, both enzymes are up-regulated in the brain of late-onset AD patients. In light of its role in pathogenesis of the diseae, BACE1 is an active target for AD translational research. Unfortunately, biochemical design of BACE1 inhibitors has proven to be challenging due to the rather large size of the catalytic pocket of the enzyme. Therefore, approaches that affect expression levels rather than catalytic activity of BACE1 are being actively sought. With this in mind, we successfully developed an in vitro assay to monitor ATase1 and ATase2 activity and conducted a High Throughput Screen (HTS) of a library of 14,400 compounds. The screen resulted in the identification of novel biochemical inhibitors of ATase1 and ATase2 that significantly reduced the levels of BACE1 and the generation of A in cellular systems. Finally, we successfully completed the necessary physical/chemical (pre-formulation) characterization and formulation development and initiated animal testing of one successful compound. The initial results show that our compound: (i) is able to cross the brain-blood-barrier (BBB) and reach the Central Nervous System (CNS) with high efficiency; (ii) reduce both BACE1 and A levels in the brain; and (iii) prevent the synaptic deficits that characterize the early AD-like pathology of the mice. HYPOTHESIS: Our central hypothesis is that functional characterization of the biological roles of ATase1 and ATase2 will help us understand the molecular mechanisms involved with the pathogenesis of AD, and that biochemical inhibitors of ATase1 and ATase2 can potentially serve to prevent or delay AD dementia. STUDY DESIGN: Specific Aim 1 will identify the molecular mechanisms that regulate the biological functions of ATase1 and ATase2 in the brain. This Aim will use a combination of in vitro, ex vivo and in vivo approaches. Specifically, we will use chromatin immunoprecipitation, DNA:protein pull-down and luciferase reporter assays to identify the transcriptional mechanism(s) responsible for the activation of ATase1 and ATase2 expression during aging and AD (Aim 1A). Mass spectrometry, site-directed mutagenesis, and a combination of biochemical and biophysical approaches will instead be used to determine the functional implications of the on-off switch that regulates ATase1 activity (Aim 1B). The physiologic and pathologic implications of the in vitro studies will be determined by using cell lines, primary neurons, post-mortem AD brain tissue and animal models of aging. Specific Aim 2 will assess whether biochemical inhibitors of ATase1 and ATase2 can serve to prevent or delay AD dementia in animal models of the disease. Animals will undergo behavioral and cognitive assessment. Post-mortem sections of the brain will be used for electrophysiological analysis of synaptic functions as well as comprehensive histological and biochemical assessment (Aim 2A). Finally, a combination of biochemical, biophysical, and cellular approaches will be used to assess whether newly identified compounds can serve as additional inhibitory tools (Aim 2B).